Patient and Tumor Characteristics
The study cohort comprised 129 patients, all diagnosed with primary HNSCC. The clinical pathological characteristics are presented in Table 1. Most cases were localized in the oral cavity and oropharynx (n=42 and n=46, respectively). 16 patients suffered from hypopharyngeal cancer, 22 had a laryngeal cancer and in 3 cases, regional lymph node metastases were obtained. Approximately half of the patients were smokers (53.1 %, ≥ 10 py) and one third without critical alcohol consumption (33.1 %). One third of the tumors (32.7 %) were p16+, because of a previous HPV infection. In 11 p16+ cases, molecular HPV testing could not be done due to poor DNA quality. These cases were classified as “HPV-like-p16+” because they were localized in the oropharynx (tonsil), without a patients’ history of noxae. One case was finally classified as p16+/HPV-. The tumors were taken from all stages (T1 – T4), with local or distant metastases in 70% and 40% of cases, respectively. From three patients with availability of tumor material from primary and meta/synchronal metastatic/recurrent disease, the staining results of primary carcinomas were included for the further analysis.
Intra- and inter-individual heterogeneity of the tumor microenvironment
The tumor microenvironment was first studied by HE-stained whole slide sections to estimate the amount and distribution of tumor-infiltrating leukocytes (TILs) (Figure 1A, B). At the invasion front, the TIL-score was significantly higher compared to the tumor center (p<0.0001, Figure 1E, left). Subtype analysis of HPVpos and HPVneg cases revealed higher infiltration levels in the former, notably in both the invasion front and the tumor center (Figure 1E, middle and right). Then, specific immunological subpopulations (T cells, macrophages, and NK cells) were quantified (Figure 1, 2, and supplementary Figure 2). Numbers of CD8+ cytotoxic T cells as well as CD68+ and CD163+ macrophages differed significantly between the invasive front and the tumor center (Figure 1F, left and Figure 2E, F, left). CD8+ T cells were higher in the HPV-associated cases, compared to their HPV-unrelated counterpart (p<0.01 (invasion front) and p<0.05 (tumor center), Figure 1F, middle and right). By contrast, macrophages (CD68+, CD163+) were similar between both subtypes (Figure 2E, F, middle and right). Amounts of CD56+ NK cells were low and showed a similar pattern in each compartment (supplementary Figure 2A, B, E, G).
Tumors in the hypopharynx had less TILs (HE, CD8+), both at the invasion front and the tumor center, respectively (p<0.05 vs. oropharynx, Figure 1G, H). Vice versa, oropharyngeal carcinomas exhibited highest values of TILs and CD8+ cells at the invasion front. Tumors of the oral cavity and the larynx had comparable TIL values (Figure 1G, H). Numbers of CD68+ and CD163+ macrophages differed significantly between different anatomical sites. Here again, hypopharyngeal cancers had the lowest numbers of infiltrating CD68+ and CD163+ macrophages in each compartment (CD68: p<0.05 vs. oral cavity and CD163: p<0.05 vs. larynx; Figure 2G, H).
Impact of HPV-Status, anatomical site, TILs, and CD8+ T cells on overall survival
Morphological examinations of HPVpos and HPVneg cancers revealed characteristic patterns in both subtypes regarding differentiation, p53 staining, and Ki-67 proliferation index (Figure 3A-H). Then, we focused on the prognostic impact of the TME on patients’ overall survival (OS) upon initial diagnosis. This analysis revealed a significant survival benefit for patients with HPV-associated HNSCC compared to their HPV-unrelated counterpart (p<0.01; Figure 3I). In the HPV-related cohort, 89% of patients received adjuvant therapy (i.e. Cisplatin, Cetuximab, and/or radiotherapy). In four patients, surgery was done without adjuvant therapy. In this small group, two patients are still alive and two deceased. The latter presented with reduced general state of health (ECOG 2 vs. ECOG 0) at diagnosis and an advanced disease stage. In the HPV-unrelated group, the choice of treatment had a minor influence on OS. Dissecting the impact of the anatomical site revealed the best OS for oral carcinomas and the worst outcome for hypopharyngeal cancer (Figure 3J). Likewise, TILs and CD8+ T cells were positively associated with OS – independent form treatment (Figure 4). For the TILs, this reached statistical significance (invasion front), and for the CD8+ T cells, the survival benefit was independent from the location within the tumors (invasion front: p<0.001; center: p<0.01, Log-rank).
PD-L1 and CMTM6-Status and its prognostic impact
While PD-L1 is an approved biomarker, we tested whether CMTM6 might have comparable prognostic value (Figures 5 and 6). First, a consensus cut-off was established to determine positivity. All samples were categorized into CMTM6 high vs. low, with a CPS≥5 defined as high. By applying this cut-off, 76.5% of all samples were CMTM6high, which is similar to the PD-L1 positivity in this cohort (CPS≥1: 74.4%). Still, PD-L1 and CMTM6 differed between individual samples (representative images are given in Figure 5C-F, Table 1). Concerning PD-L1, there was a strong correlation for CPS and TPS (Spearman r=0.779, p<0.001) as well as for CPS and ICS (Spearman r = 0.851, p<0.001) and for ICS and TPS (Spearman r = 0.616, p<0.001). By means of CPS, 74.4% of cases had a value ≥ 1 with a median CPS of 5 (Figure 5A and D). Concerning CMTM6, there was a highly significant correlation for CPS and TPS (Spearman r = 0.730, p<0.0001), and for CPS and ICS (Spearman r = 0.242, p<0.01), but not for ICS and TPS (Spearman r = -0.041). By means of CPS, 3.4% expressed a score <1 and 76.5% expressed a score ≥5. Median CPS was 25. Given the higher CMTM6 expression by CPS, cases with a CPS ≥ 5 were set positive for further analysis (whereas CPS ≥ 1 defined PD-L1 positivity). Considering CPS, there was a significant correlation between PD-L1 and CMTM6 expression (Spearman r = 0.217; supplementary Figure 3A) with an analogical staining pattern. All samples with a CMTM6 CPS ≥ 5 were PD-L1 positive. For both markers, CPS did not differ significantly between HPV-associated and HPV-unrelated HNSCCs (Figure 5A, B, middle). Likewise, the anatomical site had a minor impact on PD-L1 or CMTM6 positivity (Figure 5 A, B, right). An exception was seen for the PD-L1 CPS, which was generally lower in hypopharyngeal cancers compared to the other sites (Figure 5A, right).
Patients exhibiting a PD-L1 CPS ≥ 1 as well as the subgroup with CMTM6 CPS ≥ 5 had a significant longer OS (p<0.0001 and p<0.01, respectively; Log-rank, Figure 6A, B, E, F). Combination of both markers showed a prolonged survival in the subgroup with PD-L1 expression ≥ 1 and synchronous CMTM6 expression ≥ 5 (p<0.05; Log-rank, Figure 6C, E, F). The CMTM6high/PD-L1neg and CMTM6low/PD-L1neg subgroup showed comparable short OS (median OS: 40 months, Figure 6C, G, H, I, J). The best outcome was seen for patients with PD-L1pos/HPVpos cancers yielding a 100 % survival rate within the follow-up of >80 months (Figure 6D). Vice versa, patients with PD-L1neg/HPVneg HNSCCs had the worst outcome (median OS: 39 months). A comparable outcome was seen when CMTM6 was considered (supplementary Figure 3B).
CMTM6 CPS correlated significantly with the number of TILs at the center (Spearman r = 0.34, p<0.0001) and PD-L1 CPS with TILs at the invasive front (Spearman r = 0.266, p<0.01) and both with CD8+ T cell Score (Spearman r = range from 0.22 to 0.34). There were no significant differences of PD-L1 or CMTM6 expression between the HPVpos or HPVneg cases although the latter tended to be more PD-L1 positive (p= 0.07; Fishers-exact test).
Taking CD8+ T cells as additional predictive biomarker (supplementary Figure 3C), patients with CMTM6high tumors and a CD8+ T cell score >1 showed the best OS (supplementary Figure 3D).
Significance of TAMs, NK cells, and DKK1 expression
Neither the amount of CD163+and CD68+ macrophages, nor numbers of CD56+ NK cells showed any significant influence on OS (Figure 7, supplementary Figure 2). There was a highly significant correlation between CD68+- and CD163+-TAMs (Spearman r>0.80 for both locations, p<0.0001) with analogical staining patterns in the examined samples. Twelve patients having a CD68/CD163-ratio >1 within the tumor center showed a significantly longer OS than the patients without dominant M1-polarized macrophages (Figure 7C). Within the CD56 stained samples, 10% and 5% had a score ≥ 3, respectively in each compartment. In the DKK1-stained subgroup, by means of our evaluation method, no clinical pathological relevance of DKK1 was identifiable with 71% of samples with DKK1 positivity on tumor cells (Supplementary Figure 2F, H). DKK1 positive carcinomas showed significantly more DKK1 positive cells in the surrounding stroma than DKK1-negative specimens (p = 0.0002; Fisher-exact test).